Ultradian corticosterone pulses balance glutamatergic transmission and synaptic plasticity

Significance

A pulse of the adrenal hormone corticosterone (CORT) changes hippocampal glutamate transmission for many hours. CORT is normally released in hourly pulses, with a steeply rising amplitude just before awakening. How organisms can be prepared for imminent danger if the first high-amplitude pulse of CORT would lastingly change glutamate transmission—thus potentially deadlocking the system—has remained an enigma. We show that exposure of hippocampal cells to a second high-amplitude CORT pulse completely normalizes all aspects of glutamate transmission (including synaptic plasticity), thus lifting the potential deadlock caused by a first pulse. This ensures that the system remains fully responsive to any stressful event that requires encoding of information, an important principle that promotes survival of individuals.

Abstract

The rodent adrenal hormone corticosterone (CORT) reaches the brain in hourly ultradian pulses, with a steep rise in amplitude before awakening. The impact of a single CORT pulse on glutamatergic transmission is well documented, but it remains poorly understood how consecutive pulses impact on glutamate receptor trafficking and synaptic plasticity. By using high-resolution imaging and electrophysiological approaches, we report that a single pulse of CORT to hippocampal networks causes synaptic enrichment of glutamate receptors and increased responses to spontaneously released glutamatergic vesicles, collectively abrogating the ability to subsequently induce synaptic long-term potentiation. Strikingly, a second pulse of CORT one hour after the first—mimicking ultradian pulses—completely normalizes all aspects of glutamate transmission investigated, restoring the plastic range of the synapse. The effect of the second pulse is precisely timed and depends on a nongenomic glucocorticoid receptor-dependent pathway. This normalizing effect through a sequence of CORT pulses—as seen around awakening—may ensure that hippocampal glutamatergic synapses remain fully responsive and able to encode new stress-related information when daily activities start.

The rodent stress hormone corticosterone (CORT) is synthesized in the adrenal glands. Through the circulation, it reaches the brain, where it binds to intracellular receptors, i.e., the high-affinity mineralocorticoid receptor (MR) and lower affinity glucocorticoid receptor (GR) (1). These receptors are enriched in the hippocampus and act as transcriptional regulators (2). Intracellular MRs are already considerably occupied with low (i.e., nonstress) levels of CORT, whereas GRs become substantially bound only when corticosteroid levels increase (1).

After stress, hormone levels slowly increase and normalize after 2 h as a result of negative feedback actions in the pituitary and hypothalamus (3). Corticosteroid levels also show an endogenous circadian variation, which in fact is carried by brief hourly (ultradian) pulses (4). Pulse amplitudes steeply rise some hours before awakening and then slowly diminish again (5). The functional relevance of ultradian pulses is only starting to be understood; recent evidence suggests that pulses are necessary to maintain optimal transcriptional activity of GRs (4–8), thus coordinating essential bodily functions in preparation of the active phase.

It is well known that, through DNA binding and transcriptional regulation, GRs slowly and persistently alter glutamatergic transmission in the hippocampus (9–11). A brief CORT pulse increases the surface expression and synaptic localization of AMPA receptor (AMPAR) subunits (10) and increases the amplitude of miniature excitatory postsynaptic currents (mEPSCs) (11)—the postsynaptic response to a spontaneously released glutamate-containing vesicle. CORT-dependent signaling is thought to (partly) overlap with pathways giving rise to long-term potentiation (LTP) (12, 13). In agreement, administration of CORT occludes the subsequent induction of chemically (10) and electrically evoked LTP (9, 14). In line with GRs acting as transcriptional regulators, these effects become apparent ∼1 h after CORT administration and last for many hours. We here examined how glutamatergic transmission in the hippocampus is affected when cells are exposed to a second pulse of CORT 1 h after the first, mimicking the ultradian pattern of endogenous CORT release (6).

Results

Consecutive CORT Pulses Normalize AMPAR Surface Dynamics and Synaptic Content.

Previously, we showed in hippocampal neurons that a single pulse of CORT drastically increases the surface diffusion and synaptic content of GluA2- and GluA1-containing AMPARs several hours later (10). This CORT-induced synaptic potentiation of AMPARs was prevented by selectively and artificially blocking AMPAR surface diffusion. Furthermore, the increased surface trafficking and synaptic accumulation of AMPARs after CORT exposure likely contributed to the occlusion of classical synaptic LTP (10, 13). To investigate the impact of CORT pulsatility, we first examined how two 10-min pulses of CORT (100 nM) with an interval of 1 h affect the surface dynamics of AMPARs. For this, we took advantage of the single particle tracking approach to monitor individual particle/receptor complexes in hippocampal cultured neurons (Fig. 1A). Hippocampal networks were exposed to one or two pulses, and single nanoparticle imaging was performed 120 min after onset of the first exposure (Fig. 1B). Consistent with previous results (10), a single CORT pulse significantly increased the surface diffusion of GluA2–AMPAR (Fig. 1 C and D). Strikingly, the introduction of a second pulse of CORT—i.e., 60 min after the first—abolished this effect, yielding a surface diffusion comparable to the vehicle control situation (Fig. 1 C and D). Of note, the second pulse did not simply delay the CORT-induced AMPAR surface diffusion increase, as the dynamics measured 120 min after the second pulse (at t 180 min) were undistinguishable from the one measured 60 min after two pulses at t 120 min (value at t 180 min GluA2–AMPAR surface diffusion: median, 0.092 µm²/s; interquartile range, 4.10⁻³ to 0.14 µm²/s; n = 670 trajectories; P > 0.05 vs. CORT–CORT diffusion at 120 min; Fig. 1). The diffusion changes observed after one or two CORT pulses at t 120 min appear to be mostly caused by a change in the mobile fraction (diffusion > 0.005 µm²/s), as control (54%) and two-pulse (47%) values were lower than the fraction seen after one pulse (76%). Together, to our knowledge, these data provide the first evidence that consecutive pulses of CORT restore the surface trafficking of AMPARs toward a basal range.

Fig. 1.

Consecutive CORT pulses normalize AMPAR surface distribution and synaptic content. (A) Surface GluA2 was labeled with a single quantum dot (QD)-antibody complex (Left), allowing single particle tracking. (Right) Representative trajectories (yellow lines; 40-s duration imaging; 20-Hz acquisition rate) in cultured hippocampal neurons (15 d in vitro). (Scale bar: 5 µm.) (B) Schematic representation of the experimental protocol. Neurons were exposed to one pulse of 100 nM CORT (or vehicle) for 10 min or two consecutive 100 nM CORT pulses (or vehicle) with an interval of 60 min. In all experiments, surface AMPAR trafficking was assessed at 120 min relative to the onset of the first CORT application. (C) Representative single GluA2–AMPAR trajectories in the different experimental paradigms described in B. Note the increased lateral diffusion 120 min after a single pulse, an up-regulation not observed in presence of two pulses (100–100 nM CORT). (Scale bar: 500 nm.) (D) Distribution (median ± 25–75% interquartile range) of the instantaneous diffusion coefficients of surface GluA2–AMPAR, measured 120 min after one pulse of 100 nM CORT or two consecutive 100 nM CORT pulses (or vehicle). The diffusion coefficient was significantly increased after a single CORT pulse (0′; n = 2,790 trajectories; 100 nM CORT, n = 916; 100–100 nM CORT, n = 449; vehicle–vehicle, n = 452; Kruskal–Wallis test, P < 0.05; post hoc tests, **P < 0.01, 0′ control vs. 100 nM CORT; P > 0.05, 100 nM CORT vs. other conditions). (E) Single QDs (black dots) can be located with a high pointing accuracy in a given membrane compartment, such as the synapse (syn., gray area). (Left) Examples of 500 frame stacks obtained while tracking down single GluA2–AMPAR/QD complexes. Those 500 locations are then projected on a single image, providing the successive positions of this receptor/particle complex in the various experimental paradigms. (Right) Relative fraction of synaptic GluA2–AMPAR/QD particles (control, n = 28 neuronal fields; 100 nM CORT, n = 19; 100–100 nM CORT, n = 27; Kruskal–Wallis test, P < 0.05; post hoc tests, **P < 0.01, control vs. 100 nM CORT or 100 nM CORT vs. 100–100 nM CORT; P > 0.05, control vs. 100–100 nM CORT). (F) The PSD area was quantified by using immunocytochemical staining of Homer 1c or Shank proteins (control, n = 315 PSDs; 100 nM CORT, n = 259; 100–100 nM CORT, n = 212; one-way ANOVA test, P < 0.05; *P < 0.05, control vs. 100–100 nM CORT or 100 nM CORT vs. 100–100 nM CORT).

We next investigated the impact of consecutive CORT pulses on synaptic AMPAR signaling by using high-resolution imaging and electrophysiological means. First, the single particle tracking approach allows to point, with subwavelength accuracy (∼20 nm resolution), the location of tagged-receptors in specific membrane compartments, such as the synapse. Based on that, we estimated that, on average, fewer than 10% of the labeled AMPARs are present in the synapse under control conditions. This value significantly increased after exposure to a single CORT pulse whereas it was comparable to baseline after two pulses (Fig. 1E), indicating that the second pulse reverses or normalizes the CORT-induced GluA2-AMPAR synaptic accumulation caused by the first pulse. A similar effect was observed when we quantified the area of the postsynaptic density (PSD) of synapses exposed to a single or two CORT pulses (Fig. 1F). Of note, the increase in GluA2–AMPAR synaptic fraction observed after one pulse (+304%) was larger than the increase of the PSD area (122%), suggesting that the altered AMPAR content is an active process that cannot solely be explained by a change in PSD area. Together, these data demonstrate that a second pulse of CORT (60 min after the first pulse) reverses or prevents the synaptic accumulation of glutamate synapses observed 120 min after a single pulse.

We predicted that functional indices of glutamatergic transmission would follow a similar pattern, i.e., that signals via AMPAR subunits would be enhanced 2 h after a single pulse but normalized when tested after two consecutive pulses. In CA1 pyramidal neurons, recorded in slices from 2-mo-old mice, we first replicated that, ∼120 min after a single brief (10 min) pulse of CORT, the mEPSC amplitude was significantly increased (vehicle, 14.3 ± 0.6, n = 9; CORT, 18.4 ± 1.3, n = 7; P < 0.01; Fig. 2). As predicted, such increase was not seen in a second series of experiments in which the 2-h delay after the first pulse was interrupted by another CORT pulse (60-min interval between pulses; vehicle, 16.4 ± 1.0, n = 7; CORT, 16.9 ± 1.0, n = 8; P = 0.73; Fig. 2). In none of the conditions did CORT treatment affect mEPSC frequency (all P > 0.05; Table S1). Altogether, these data provide direct evidence that two consecutive pulses, 60 min apart, restore the AMPAR surface dynamics and synaptic content to values close to those seen under basal conditions.

Fig. 2.

A single CORT pulse increases mEPSC amplitude, whereas two pulses do not. (A) Typical traces showing mEPSCs of a mouse CA1 hippocampal cell ∼120 min after exposure to a brief (10 min) pulse of vehicle (Left) or 100 nM CORT (Right). (B) The mEPSC amplitude was significantly enhanced in neurons recorded ∼120 min after a single pulse of CORT as opposed to vehicle (Left). By contrast, two consecutive CORT pulses, with a 1-h interval, caused no change in mEPSC amplitude compared with two vehicle pulses when neurons were recorded ∼120 min after onset of the first pulse. Data represent the mean ± SEM of the mEPSC amplitudes recorded 5–10 min after establishing the whole cell configuration. To allow easy comparison, we normalized the effects of CORT exposure to those of vehicle in both experiments (see main text for absolute values). *P < 0.01, unpaired t test.

CORT Pulses Restore the Plastic Range of Hippocampal Glutamate Synapses.

Earlier studies showed that exposure of hippocampal slices to a single pulse of CORT impairs the induction of LTP in subsequent hours (9, 14). This was interpreted as a CORT-induced occlusion of pathways necessary for the development of LTP (12, 13). We therefore examined how synaptic plasticity is affected by consecutive pulses. First, we tested the impact of consecutive pulses on LTP expression in hippocampal cultured neurons by using a previously described chemical LTP (cLTP) induction protocol (10) and measuring the synaptic content (synapses were identified by Homer 1c detection) of AMPARs through live immunocytochemical staining (Fig. 3A). We focused our attention on GluA1–AMPAR because of their primary and well-characterized recruitment to synapses following LTP (15). Application of a single pulse of CORT prevented the cLTP-induced GluA1–AMPAR synaptic recruitment, as previously described (10) (Fig. 3 B and C).However, when neurons were exposed to two consecutive pulses, full-blown synaptic recruitment was observed 120 min after the first pulse (Fig. 3B), indicating that the second pulse restores the potentiation range of the glutamate synapses. Consistent with the aforementioned electrophysiological data, two consecutive pulses of CORT maintained the synaptic content of GluA1–AMPAR close to basal values (Fig. 3C). Together, these data indicate that a second pulse of CORT normalizes the potentiation range of glutamate synapses, which was occluded by the first pulse.

Fig. 3.

Consecutive CORT pulses restore cLTP-induced AMPAR potentiation. (A) (Left) Representative dendritic fragment of hippocampal neurons expressing GluA1–SEP AMPAR. (Scale bar: 1 µm.) (Right) Enlargement of a synaptic GluA1–SEP cluster, with the postsynaptic area labeled by the detection of Homer-1c (dashed black line). (B) (Left, Center) GluA1–SEP dendritic fragments from neurons under basal condition or after the application of a cLTP protocol with or without the presence of CORT pulses [protocols (Left): control cLTP, 100 nM CORT cLTP, 100–100 nM CORT cLTP, or 100–100 nM CORT]. The pseudocolor GluA1–SEP subunit representation shows the different intensity levels of the GluA1–SEP staining. (Scale bar: 1 µm.) (Right) Enlargement of a GluA1–SEP cluster. (Scale bar: 500 nm.) Note that cLTP fails to potentiate GluA1–SEP fluorescence after a single CORT pulse. (C) Normalized measures of GluA1–SEP clusters intensity compared with baseline (n = 3,495 clusters). The cLTP protocol induced an increased GluA1–SEP expression in control condition (one-way ANOVA test, P < 0.05; control cLTP, n = 2,619 clusters; **P < 0.01) and after consecutive CORT pulses (100–100 nM CORT cLTP, n = 1,458 clusters; **P < 0.01), whereas GluA1–SEP fluorescence remained unchanged after a single pulse (100 nM CORT cLTP, n = 4,372 clusters).

Hippocampal cultured cells are not connected to their normal afferent and efferent fibers as in the intact brain. We therefore switched to acutely prepared hippocampal slices from young adult mice and used a high-frequency paradigm that (in vehicle-treated slices) causes mild LTP in the CA1 area (Fig. 4 A1 and B). Induction of LTP was fully prevented by a single 10-min pulse of CORT applied to the slices 120 min before high-frequency stimulation [F_(2, 21) = 15.77; P < 0.001; vehicle vs. one CORT pulse; P < 0.001; Fig. 4 A1, B, and C]. However, if we applied two CORT pulses (with a 1-h interpulse interval), synaptic potentiation caused by high-frequency stimulation 120 min after the first pulse was comparable to that seen in the vehicle group (P > 0.1 vs. vehicle; Fig. 4 A2, B, and C). As 100 nM CORT is most likely a concentration not reached under physiological conditions, we also tested two pulses of 30 nM CORT. This resulted in comparable results [F_(2, 21) = 9.87; P < 0.01], i.e., suppression of LTP 120 min after a single CORT pulse (P < 0.01 vs. vehicle), but efficient LTP after two pulses (P > 0.1 vs. vehicle; Fig. 4D and Fig. S1A). We also wondered to what extent the naturally occurring 1-h interval is relevant for the observed phenomenon. As is evident from Fig. 4E and Fig. S1B, the interval is indeed important [F_(4, 29) = 12.52; P < 0.001]: no normalization of LTP was seen when the second pulse was given 10 min after the first pulse (P < 0.001 vs. vehicle), and only partial normalization was found with an interpulse interval of 30 min (P < 0.05 vs. vehicle). With intervals of 60 or 90 min, LTP induced 120 min after onset of the first pulse was comparable to that in the vehicle control group (P > 0.1 vs. vehicle). Together, these results suggest that the CORT-induced restoration of LTP is physiologically relevant and depends on the timing of the second CORT pulse.

Fig. 4.

Synaptic plasticity in hippocampal slices is restored by consecutive CORT pulses. (A1 and A2) Schematic representation of the experimental protocol. (A1) Mouse brain slices were exposed to one pulse of 100 nM CORT (or vehicle) for 10 min. (A2) Brain slices were exposed to two consecutive 100 nM CORT pulses (or vehicle) with an interval of 60 min. In both experiments, high-frequency stimulation (HFS) was applied at 120 min relative to the onset of the first CORT application. (B) HFS (10 Hz, 90 s) resulted in significant potentiation of synaptic responses at CA1 synapses in vehicle-treated brain slices (open circles). LTP was attenuated by 100 nM CORT given 120 min before HFS (gray circles). A second application of 100 nM CORT 60 min after the first pulse reversed this effect (black circles). (Upper) Representative individual fEPSP traces for the vehicle group taken during baseline or ∼20 min after HFS. (Scale bars: horizontal, 5 ms; vertical, 0.5 mV.) (C) Averaged mean values during the 60-min posttetanic recording period indicate significantly lower LTP in slices treated 120 min earlier with one 100 nM CORT pulse (gray bar) compared with vehicle (open bar). A second 100-nM CORT pulse applied 60 min after the first yielded LTP comparable to that seen in the vehicle group (black bar). (Upper) Experimental paradigm (see also A1 and A2).The second CORT pulse in between brackets was applied for only the group receiving two CORT pulses (100–100 nM CORT). All groups, n = 8. (D) Similar results were found with lower, physiologically more relevant CORT concentrations (30 nM). (Upper) Schematic representation of the experimental protocol in which slices were exposed to one or two 30 nM CORT pulses with an interval of 60 min. The second CORT pulse between brackets was applied for only the group receiving two CORT pulses (30–30 nM CORT). HFS was applied 120 min after the start of the first pulse (vehicle and 30 nM CORT, n = 8; 30–30 nM CORT, n = 6). (E) Variations in the duration of the interval between the two 100-nM CORT pulses. Normalization of LTP was seen with 60- and 90-min intervals but not with shorter (10 or 30 min) intervals between the two CORT pulses. (Upper) Schematic representation of the experimental protocol in which the delay between application of the two 100-nM CORT pulses was varied between 10 and 90 min. In all cases, HFS was applied 120 after the start of the first pulse. Vehicle and 60 min delay group (100–100 nM CORT), n = 8; all other groups, n = 6. Values indicate group means ± SEM (***P < 0.001, **P < 0.01, and *P < 0.05).

CORT Pulses’ Effects on the Plastic Range Are Nongenomic and GR-Dependent.

To better understand the mechanism contributing to normalization of LTP as seen after two pulses of CORT, we first questioned whether the second pulse reversed or prevented the effect of the first pulse. Therefore, we tested if suppression of LTP after a single pulse was already seen after 60 min, i.e., at the time point at which the second CORT pulse normally would be delivered. As shown in Fig. 5A and Fig. S2A, this was indeed the case [F_(4, 30) = 10.75; P < 0.001; P < 0.001 vs. vehicle). Additionally, to exclude time-dependency of CORT effects, we tested for LTP 120 min after the second pulse (at t 180 min). At this time point, LTP was efficiently induced, suggesting that the second pulse lastingly reverses or compensates for the consequences of the first CORT application (P < 0.05 vs. 100 nM CORT; Fig. 5A and Fig. 2A). The results from the experiment in which two pulses were applied with a 90-min interval (leaving only 20 min between the end of the second pulse and LTP induction; Fig. 4E) suggest that this reversal or compensation develops rapidly, in a time frame that seems incompatible with the classic genomic pathway. Such nongenomic signaling by CORT has indeed been described in recent years (16–18). In the hippocampus, nongenomic signaling so far was found to involve MRs (16), but, in other areas of the brain (e.g., the hypothalamus or amygdala), rapid effects through GRs have been described (17, 18). To determine the receptor mediating the reversal or compensation, we applied the second pulse of CORT in the presence of an MR or GR antagonist. As is clear from Fig. 5B and Fig. S2B, we observed significant differences [F_(4, 34) = 13.68; P < 0.001] between the treatment groups. The MR antagonist spironolactone was entirely ineffective in blocking the effect of CORT (P > 0.1 vs. vehicle), whereas the GR antagonist mifepristone fully prevented the normalizing effect of the second CORT pulse (P < 0.001 vs. vehicle), supporting involvement of GRs. Nongenomic, as opposed to genomic, actions of CORT can be mimicked by the membrane impermeable conjugate of CORT to BSA (CORT–BSA). In agreement with a nongenomic pathway, normalization of LTP was replicated when slices were exposed to CORT–BSA, delivered 60 min after a first pulse of CORT [F_(4, 33) = 4.16; P < 0.01; P > 0.1 vs. vehicle or 100–100 nM CORT; Fig. 5C and Fig. S2C]; in agreement with the genomic pathway underlying the response to the first pulse, a single pulse of CORT–BSA was unable to suppress LTP induced 120 min later (P > 0.1 vs. vehicle). Finally, we reasoned that, if the second pulse of CORT would compensate for (i.e., act in the opposite direction) rather than reverse the response to the first pulse, a sequence of two pulses of CORT–BSA might result in enhanced LTP compared with the effect seen with two pulses of CORT. Although LTP levels were, on average, slightly increased after two pulses of CORT–BSA, especially directly after high frequency stimulation, we did not observe a significant effect [F_(3, 27) = 14.67; P < 0.001; P > 0.1 for CORT–BSA/CORT–BSA vs. vehicle or 100 nM CORT–CORT; Fig. 5D and Fig. S2C]. Together, these experiments indicate that the normalizing effect of consecutive CORT pulses is mediated by a noncanonical membrane GR-dependent signaling pathway.

Fig. 5.

Normalization of LTP depends on nongenomic GR-dependent pathway. (A) Averaged mean values during the 60-min posttetanic recording period. Application of one 100 nM CORT pulse at the moment of the second CORT pulse (bar with white dashed stripes) still attenuated LTP compared with vehicle-treated slices (open bar) in a similar way as 120 min after a single CORT pulse (gray bar). HFS applied 120 min after the onset of the second CORT pulse (at t 180 min) very effectively induced LTP (bar with black dashed stripes). (Upper) Experimental paradigm. All groups, n = 8; second pulse CORT group, n = 6; 2 h delay, n = 5. (B) The presence of spironolactone during the second CORT administration (black dashed bar) did not affect synaptic potentiation, whereas mifepristone (gray dashed bar, far right) significantly attenuated the level of LTP. (Upper) Schematic representation of the experimental protocol. Coincubation with 100 nM spironolactone or 500 nM mifepristone started 20 min before the application of the second 100-nM CORT pulse and coterminated with the second pulse. HFS was applied 120 min after the start of the first pulse. Spironolactone, n = 7; all other groups, n = 8. (C) When CORT–BSA (dashed black bar, far right) was delivered during the second pulse instead of CORT (black bar), synaptic plasticity was normalized, and fully comparable to what was seen in slices treated only with vehicle (open bar). A single pulse of CORT–BSA did not affect synaptic plasticity (dashed open bar), different from the reduction seen with a single CORT pulse (gray bar). (Upper) CORT–BSA was applied as a single pulse (100 nM CORT–BSA) or as a second pulse after a CORT pulse (second pulse CORT–BSA). HFS was applied 120 min after the start of the first pulse. Second pulse CORT-BSA and 100 nM CORT–BSA, n = 7; all other groups, n = 8. (D) Two consecutive pulses of 100 nM CORT–BSA (dashed black bar) did not significantly increase LTP levels compared with vehicle-treated slices (open bar). (Upper) Two pulses of 100 nM CORT–BSA were administered with an interval of 60 min. HFS was applied 120 min after the start of the first pulse (100–100 nM CORT–BSA, n = 7; all other groups, n = 8). Values indicate group means ± SEM (*P < 0.05, **P < 0.01, and ***P < 0.001). Note that the vehicle, single-pulse CORT, and double-pulse CORT groups shown in this figure are based on the same data as Fig. 4 to allow comparisons.

Discussion

A single pulse of CORT facilitates glutamatergic transmission in hippocampal cells (9–11, 13), a condition that is associated with an impaired ability to subsequently induce synaptic potentiation (12, 13). This has been interpreted earlier as a means to promote the encoding of stress-related information, by protecting the encoding process from interfering input reaching the same synapses at a later point in time (12). Although this may be beneficial when CORT is released as part of a stress response, it may deadlock the system when CORT is increased as a result of endogenous circadian variations: this would fully prevent encoding of information for many hours after the first high-amplitude ultradian CORT pulse before awakening.

We show that a second pulse of CORT, hitting the cells at a time point when the effects of the first pulse have already developed, can fully normalize all investigated aspects of glutamatergic transmission in the hippocampus and restore responsiveness to high-frequency dependent encoding. The system does so by triggering an entirely different signaling pathway after the second pulse than the one involved in the first pulse. Although, in the absence of CORT, surface expression of corticosteroid receptors is apparently low (because CORT–BSA was ineffective in mimicking the effect of CORT in the single-pulse paradigm), the first pulse of CORT may trigger surface localization of GRs, thus priming the system to a nongenomic effect that effectively reverses the genomic suppression of LTP.

The observation that limbic cells respond differently to a second pulse of CORT than to the first is not unprecedented. Recently, we reported that a single pulse of CORT quickly but persistently increases mEPSC frequency in amygdala cells (18); through this persistent effect, amygdala cell characteristics are slowly changed such that they respond in an opposite manner to a second pulse arriving >1 h after the first. Here we show that, in the hippocampus, metaplastic responses can be observed not only at the level of mEPSC amplitude, but also with respect to AMPAR trafficking and synaptic plasticity.

The selected CORT application paradigm was based on earlier studies (e.g., ref. 6) and mimics as closely as possible the natural pulsatile pattern with gradually rising/falling flanks and very low CORT levels during the interpulse interval (4) (Fig. S3). It therefore seems unlikely that our observations are a mere product of an artificial “on/off” paradigm. Although our reduced preparations—cultured cells and brain slices—allow a straightforward interpretation of the results, we are fully aware that they lack the complex connectivity of the brain and connections with peripheral organs. It would thus be interesting and important to see if similar phenomena occur in the intact organism. Unfortunately, this is currently not feasible at the single-cell level because of technical issues.

Most of the experiments described here were performed with 100 nM CORT because earlier studies have shown very clear actions with this concentration. Most likely, this is a supraphysiological concentration (19). The fact that we also saw normalization of LTP with two pulses of 30 nM CORT supports physiological relevance, but future experiments would need to explore the concentration dependency in greater detail. Similarly, it would be of interest to follow metaplastic changes for more than the current 2-h range. Finally, several studies have shown that nuclear GR signaling—e.g., translocation, binding, and transcription of target genes—normalizes to baseline levels between pulses (4, 6–8). It is therefore of great interest to resolve in detail if and particularly how a first pulse of CORT could lead to enhanced surface expression of GRs and which signaling pathways underlie the reversal exerted by the second pulse on processes activated by the first pulse. In that respect, additional experiments that interfere with transcriptional and/or translational regulation by CORT would strengthen the notion of nongenomic signaling regarding the latter, which is presently based only on our results with CORT–BSA.

In summary, we show that pulsatile exposure to CORT has the potential to maintain glutamatergic transmission in the hippocampal CA1 area at a stable level, preventing a “fixed” situation that may arise after a single pulse. This maintenance is in line with the observation that pulsatile CORT release is important to maintain transcriptional activity via GRs at an optimal level (4–8). If so, pulsatile CORT exposure would prepare the organism in a flexible manner to upcoming challenges at the start of the active period of the day.

Materials and Methods

Drugs.

CORT (30 or 100 nM; first dissolved in 90% (vol/vol) ethanol and next diluted to the final concentration, containing a maximum of 0.009% ethanol) was administered in pulses of 10 min, which effectively cause an increasing concentration during this period, followed by a slow decrease in the subsequent 10-min period (Fig. S3). This paradigm was previously applied to mimic ultradian pulses in vitro (6). To determine the receptor involved, we applied the selective MR antagonist spironolactone (100 nM; first dissolved in chloroform and next diluted to the final concentration, containing 0.002% chloroform) or the GR antagonist mifepristone (500 nM; in H₂O to which 5 N HCl was added in an amount just sufficient to dissolve the antagonist, and then diluted to the final concentration). All drugs were obtained from Sigma-Aldrich. To study the involvement of putative membrane-bound corticosteroid receptors, we used CORT–BSA conjugate (100 nM; first dissolved in H₂O and then diluted to the final concentration; Sigma-Aldrich).

Hippocampal Cell Culture, Protein Expression, Immunocytochemistry, and cLTP.

Cultures of hippocampal neurons were prepared from embryonic day 18 Sprague–Dawley rats following a previously described method (20). Briefly, cells were plated at a density of 200–300 × 10³ cells per dish on poly-lysine–precoated coverslips. Mixed cultures of neurons and glial cells were layered on coverslips and maintained in a 3% (vol/vol) serum containing Neurobasal medium. This medium was replaced after 4 d in vitro by a serum-free Neurobasal medium, and cultures were kept at 37 °C in 5% (vol/vol) CO₂.

Pulses of CORT (20 min total; 10 min exposure and 10 min washout; comparable dynamics and kinetics as shown in Fig. S3) were applied to the neuronal coverslips through a peristaltic pump (2 mL/min for CORT pulse, 1 mL/min between pulses). For live immunocytochemical staining, neurons were transfected with GluA1–super ecliptic pHluorin at 8–10 d in vitro by using the Effecten transfection kit (Qiagen) following the provider’s protocol. Surface GluA1–AMPARs were specifically stained by using a monoclonal anti-GFP antibody (1:500; Roche) for 15 min on live neurons at 37 °C in culture medium. Briefly, neurons were then fixed with 4% (wt/vol) paraformaldehyde/4% (wt/vol) sucrose for 15 min, washed, and incubated with secondary anti-mouse Alexa 488 antibodies (1:600, 30 min). To label postsynaptic areas, neurons were permeabilized by using 0.1% Triton X-100, incubated with a primary guinea pig polyclonal anti-Homer 1c antibody (1:500, 30 min; Synaptic Systems) or shank antibody (rabbit polyclonal antibody; 1:1,000, 1 h; Abcam), and finally incubated with secondary anti-guinea pig Alexa 568 antibodies (1:600, 30 min; Molecular Probes) or anti-rabbit Alexa 488 antibodies (1:600, 30 min; Molecular Probes). Neurons were washed, mounted, and preparations were kept at 4 °C until observation. For the quantification of surface AMPAR staining within individual synapses, Homer 1c staining served as a mask filter to isolate surface GluA1 subunit staining in individual Homer 1c clusters. The integrated fluorescence level over the Homer 1c cluster area was then measured for each cluster. The fluorescence analysis was performed by using imaging tools from Metamorph software (Universal Imaging), as previously described (21–23). The chemically induced potentiation protocol consisted of a bath coapplication of glycine (200 µM) and picrotoxin (5 µM) for 4 min. In all live experiments, cLTP was always applied after a period of baseline acquisition and the medium was carefully replaced by fresh equilibrated and heated medium after induction.

Single Quantum Dot Tracking.

Single particle detection and imaging were performed as previously described in detail (24, 25). In-depth information is given in SI Materials and Methods.

Animals.

Male C57BL/6 mice (∼7–8 wk of age at arrival; Harlan) were group-housed at a 12-h light/dark schedule (lights on at 0700 hours) with food and water provided ad libitum. After an acclimatization period of ∼1–2 wk, mice (one at a time) entered the experiment. Mice were decapitated early in the morning when endogenously circulating plasma levels are still low. All experiments were approved by the animal ethical commission of Utrecht University, and all efforts were made to minimize suffering of the animals.

Electrophysiology.

Early in the morning, the mouse was decapitated and the brain was rapidly dissected and placed in ice-cold artificial cerebrospinal fluid (aCSF) containing (in mM) 120 NaCl, 3.5 KCl, 1.3 MgSO₄, 1.25 NaH₂PO₄, 2.5 CaCl₂, 10 glucose, and 25 NaHCO₃, and continuously gassed [mixture of 95% (vol/vol) O₂/5% (vol/vol) CO₂]. Next, dorsal hippocampal slices (350 μm) were made by using a Vibratome (VT 1000S; Leica) and stored in aCSF at room temperature for >1 h before recording commenced at a bath temperature of 30–32 °C (16).

For patch-clamp electrophysiology, one slice at a time was transferred to the recording chamber mounted on an upright microscope (Axioskop 2 FS plus; Zeiss) with differential interference contrast and a water immersion objective (×40) to identify the cells. Slices were continuously perfused (flow rate, 1.5 mL/min) with warm aCSF (temperature, 30 °C; pH 7.4) containing TTX (0.5 µM; Latoxan) to block sodium channels and bicuculline (50 µM; Enzo) to block GABA_a receptors. Slices were exposed to a single 10-min CORT (100 nM; or vehicle as control) pulse or two consecutive pulses (of CORT or vehicle) with a 60-min interval. Unless stated otherwise, all recordings were performed ∼2 h after onset of the first pulse.

Patch pipettes (borosilicate glass pipettes; inner diameter, 0.86 mm; outer diameter, 1.5 mm; Hilgenberg) were pulled on a Sutter micropipette puller (resistance; 3–6 MΩ).The intracellular solution contained (in mM): 120 Cs methane sulfonate, 17.5 CsCl, 10 Hepes, 2 MgATP, 0.1 NaGTP, 5 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), pH 7.4, adjusted with CsOH. BAPTA was obtained from Molecular Probes, and all other chemicals were obtained from Sigma-Aldrich. An Axopatch 200B amplifier (Axon Instruments) was used for whole-cell recordings, operating in the voltage-clamp mode. The patch-clamp amplifier was interfaced to a computer via a Digidata (type 1200; Axon Instruments) analog-to-digital converter. Data acquisition and storage was done by using PClamp (version 9.2).

Holding potential was −70 mV. The liquid junction potential caused a shift of 8 mV at most, for which we did not compensate. Recordings with an uncompensated series resistance of <2.5 times the pipette resistance were accepted for analysis. In view of the small current amplitudes, the recordings were not corrected for series resistance. Currents were identified as mEPSCs when the rise time was faster than the decay time; mEPSC frequency and peak amplitude were determined.

Field excitatory postsynaptic potentials (fEPSPs) were recorded in the Schaffer collateral-CA1 pathway of coronal mouse brain slices as described previously (26). Briefly, a bipolar stimulation electrode (60 μm stainless-steel wires insulated except for the tip) was placed on the Schaffer collaterals, and glass recording pipettes (filled with aCSF; 2–5-MΩ impedance) were positioned in the CA1 stratum radiatum. At the start of each experiment, an input–output curve was established to record the slope of the fEPSP, from which maximal and half-maximal slope as well as the corresponding maximal and half-maximal stimulus intensity were determined. The half-maximal stimulus intensity that was calculated was used throughout the remainder of the recording session. For each experimental group, baseline synaptic transmission was recorded with a frequency of 0.033 Hz (0.15 ms) for 10 min. Thereafter, repetitive tetanic stimulations (10 Hz; 90 s) were applied, after which recordings proceeded for another 60 min at a frequency of 0.033 Hz; this stimulation paradigm is very sensitive to the effects of CORT (26). Two consecutive traces were averaged to represent the mean per minute. Data were acquired, stored, and analyzed by using Signal 2.16 (Cambridge 159 Electronic Design).

Data Analysis.

Because data of diffusion coefficients are not normally distributed, comparisons between groups for instantaneous diffusion coefficients were performed by using a Mann–Whitney test (pair comparison) or Kruskal–Wallis test followed by Dunn multiple comparison test (group comparison). All other group comparisons for live cell imaging or single-cell electrophysiology were performed by using parametric statistical tests, Student t test (unpaired or paired comparison as appropriate), ANOVA followed by Newman–Keuls multiple comparison test (group comparison), or Kolmogorov–Smirnov test (distribution comparison). Significance levels were defined at P < 0.05, P < 0.01, or P < 0.001.

For mEPSC properties, data for both series (a single CORT/vehicle pulse or two CORT/vehicle pulses) were compared with an unpaired Student t test. For the fEPSP recordings, all statistical analyses were done with SPSS 21.0. Group values are expressed as mean ± SEM. A one-way ANOVA was used to assess group differences in the level of potentiation. Where applicable, Tukey post hoc tests were used. Student paired t tests was used to compare within-group baseline values with the level of LTP. P values <0.05 were considered to indicate a significant difference.